During the last years, there was great progress in the field of genetic engineering, and some systems using genetically manipulated microorganisms, especially strains of the enterobacterium Escherichia coli and of baker's yeast (Saccharomyces cerevisiae), are now working. However, there exists a need for additional and improved systems, especially eukaryotic systems, such as yeasts, which are suitable for the economic and large-scale production of proteins in industry. At present, various yeast vectors are available for gene cloning. For the efficient expression of foreign genes in yeast structural coding sequences will have to be combined with strong yeast promoters which, advantageously, should show regulatory features which allow exogenous control of gene expression. Since efficiency of expression (product formation) is believed to be a function of and proportional to the strength of the promoter employed, workers in the field of genetic engineering pay special attention to the development of powerful promoter systems.
In vitro mutagenesis of cloned yeast genes coding for proteins and their reintroduction back into yeast cells for functional analysis have allowed the identification of various essential cis-acting promoter elements [for review see L. Guarente, Cell 36, 799(1984)]. Beginning with the elements immediately flanking the protein coding region at the 5' end of the gene these elements include:
a 5' transcribed leader region, rather A-T rich, sometimes including a CAACAACAA (or related sequence) motif, PA1 transcription initiation points, located about 40 to 60 bp (sometimes more) from the translational start codon ATG, usually pointing to a multiplicity of mRNA start sites of different strengths, PA1 a TATA box (sometimes more than one), located about 40 to 80 bp from the transcription initiation points, presumably acting as essential RNA polymerase II recognition site, PA1 upstream activation site(s) (UAS), presumptive target sites for regulatory proteins, located about 100 to 300 bp upstream from the TATA box. PA1 (1) Removal of the yeast cell wall or parts thereof. PA1 (2) Treatment of the "naked" yeast cells (spheroplasts) with the transforming DNA in the presence of PEG (polyethyleneglycol) and Ca.sup.2+ ions. PA1 (3) Regeneration of the cell wall and selection of the transformed cells in a solid layer of agar.
The UAS acts in a manner distinct from the regulatory sites found in procaryotes and resemble more the enhancer sites of the mammalian systems. Rather detailed data are available from the yeast GAL 1, GAL 7, GAL 10 cluster where a positively acting regulatory protein (GAL 4 gene product) interacts directly with the UAS of GAL 1-GAL 10 [Giniger et al., Cell 40, 767(1985)].
By fusing promoter segments encoding the UAS of GAL 1-GAL 10 in front of the TATA box of the yeast CYC 1 gene a hybrid promoter was generated whose transcription is now under the control of the UAS of GAL 1-GAL 10, i.e. it is GAL 4 gene dependent [L. Guarente et al., Proc. Natl. Acad. Sci. USA 79, 7410(1984)]. A similar construction was done by fusing promoter elements of CYC 1 and LEU2 [L. Guarente et al. Cell 36, 503(1984)]. Both of these examples include promoter elements and protein coding sequences from yeast and no evidence that these systems work also with genes foreign to yeast is available.
A recently published patent application (PCT 84/4757) describes a UAS element of the yeast PGK gene. The presence of an essential promoter element located between positions -324 and -455 (from the ATG) is shown. It is alleged that addition of this element in front of other promoters would potentiate the strength of any other yeast promoter. However, no example substantiating this allegation is given, the arguments depending entirely on negative data (destroying a promoter). It is well possible that the element is an essential part of the PGK promoter but it is doubtful whether such an element would work as part of a hybrid promoter. In addition, the UAS of PGK is not associated with a regulatory signal, i.e. it does not allow to control the expression (transcription) of the downstream coding sequence by a specific physiological signal.
Some of the promoters of glycolytic genes are induced in the presence of glucose. They can potentially be turned off if the cells are grown in a glucose-free medium. This means that yeast host cells would have to be transformed and regenerated in a medium where glucose is replaced by other carbon sources (acetate, glycerol, lactate, etc.) in order to protect the cells against a potentially harmful or lethal gene product accumulating within the cells. Since regeneration of protoplasts [A. Hinnen et al. Proc. Natl. Acad. Sci. USA 75, 1929(1978)] or of salt treated whole cells [Ito et al. J. Bacteriol. 153, 163(1983)] is generally done in a rich medium in order to allow rapid regeneration of the cells and formation of colonies, all currently used transformation protocols use glucose as a carbon source. It is expected that regeneration and recovery in a glucose-free medium works very poorly (if at all).
It is generally recognised in the art that the timing of expression must be regulated to ensure that the protein is produced at high levels only when the cell can best tolerate the large amounts of foreign proteins, i.e. outside the growth period. It is also desirable that regulation of expression does not depend on the presence or absence of the most important carbon source for microbial growth, viz. glucose. Regulable and strong promoter systems meeting these requirements for the convenient and technically applicable expression of foreign genes by yeast are scarcely known in the art. There is thus a need for the development of such promoter systems.
Surprisingly it has now been found that combination of the TATA box region of promoters controlling the expression of enzymes involved in the glycolytic pathway and generally believed to belong to the strongest promoters known at present, with upstream promoter elements of a regulable promoter the repression or derepression of which does not depend on the presence or absence of an essential component of the growth medium, such as an essential carbon or nitrogen source, leads to strong hybrid promoters meeting the main requirements imposed on technically applicable promoter systems.